Endocannabinoid System Is Involved In The Neuroprotective Effect Of Pretreatment With Nicotine Against Focal Cerebral Ischemia

Epidemiological datas have shown that nicotine may reduce the incidences of Parkinson's disease and Alzheimer's disease [1]. In recent years, further studies on nicotine in vitro experiments and in animal studies shown that nicotine could reduce the cytotoxic effect induced by N-methyl-D-aspartate receptor, delay the death of P12 cells which lack of nerve growth factor[2, 3], and improve spontaneous motor activities through againsting methamphetamine's neuronal degeneration effect[4] .These studies suggested nicotine may has a possible neuroprotective effect. Moreover, CB1 receptor antagonist, decreased nicotine self-administration and conditioned place preference in rats, chronic nicotine injections increased endocannabinoids levels in brain [5-7]. Suggest there may be an interaction between nicotine and endocannabinoid system. As the evendences that endocannabinoid system plays an neuroprotect role in the nerve damage[8] ,we suppose pretreatment with nicotine could produce rapid tolerance to focal cerebral ischemia via regulation of endocannabinoid system.Middle cerebral artery occlusion(MCAO) model was used in this study. By the way of precondition, we want to explore whether nicotine plays a neuroprotective role in the focal cerebral I/R injury, and the possible mechanism, provid new ideas for drug pretreatment in the clinical application.This study consists of four Experiments: 1. The effect of nicotine preconditioning on I/R injury in rats.;2. CB1 receptor antagonist AM251 reduced neuroprotective effect of nicotine preconditioning; 3. Observe the content changes of endocannabinoid 2-AG and AEA in brain after nicotine injection; 4. Observe the changes of endocannabinoid receptor CB1 in brain after nicotine injection by Western Blot and Immunofluorescence staining.Experiment 1: The effect of nicotine preconditioning on I/R injury in rats.Methods 30 male SD rats(280-320g)were randomly divided into 3 groups(n=10): MCAO group; Nicotine+MCAO group : Nicotine hydrogen tartrate salt solution was given 2hrs before MCAO (1.2mg/kg, intraperitoneally); TA+MCAO group: tartrate solution was given 2hrs before MCAO (0.4mg/kg, intraperitoneally). Used the method of Garcia to obtain the neurological behavioral score twenty four hours after reperfusion, then removed the brains for TTC staining to measure the percentage of infarct volume.Results Compared with MCAO group the values of NBS in the Nicotine+MCAO group were better(P<0.05), and the percentage of brain infarct volume was less(P<0.05), there were no statistical differences between MCAO group and TA+MCAO group(P>0.05)Conclusion: Nicotine pretreatment has a protective effect on cerebral I/R injury of rat. Experiment 2: CB1 receptor antagonist AM251 reduced neuroprotective effect of nicotine preconditioning.Methods 40 male SD rats(280-320g) were randomly divided into 5 groups(n=8): MCAO group, Nicotine+MCAO group: Nicotine hydrogen tartrate salt solution was given 2hrs before MCAO (1.2mg/kg, intraperitoneally); AM251+Nicotine+MCAO group: AM251 (10mg/kg, intraperitoneally) was given 30min before the injection of nicotine hydrogen tartrate salt; Vehicle+Nicotine+MCAO group: Vehicle the solvent of AM251 was given 30min before the injection of nicotine hydrogen tartrate salt; AM251+MCAO group: AM251 was given 2.5hrs before MCAO (10mg/kg, intraperitoneally). After 24hours reperfusion rats were sacrificed to assess the NBS and percentage of brain infarct volume .Results Compared with Nicotine+MCAO group the values of NBS in the AM251+Nicotine+MCAO group were lower(P<0.05), and the percentage of brain infarct volume was higher(P<0.05), there were no statistical differences between Nicotine+MCAO group and Vehicle+Nicotine+MCAO group(P>0.05). Compared with MCAO group the values of NBS in the AM251+Nicotine+MCAO group were higher(P<0.05), and the percentage of brain infarct volume was lower(P<0.05), there were no statistical differences between MCAO group and AM251+MCAO group(P>0.05)Conclusion The neuroprotection of nicotine pretreatment on cerebral I/R injury can be partially reversed by endocannabinoid receptor antagonist AM251. Experiment 3: Observe the content changes of endocannabinoid 2-AG and AEA in brain after nicotine injection.Methods 54 male SD rats(280-320g), were randomly divided into 9 groups(n=6): 0.5h TA group, 0.5h Nicotine group, 1h TA group, 1h Nicotine group, 2h TA group, 2h Nicotine group, 3h TA group, 3h Nicotine group. After intraperitoneal injection at specified time, the brains of animals in all groups were removed. Observed the changes of endocannabinoid content(2-AG and AEA) in the ipsilateral hemisphere by liquid chromatography-mass spectrometry.Results Compared with Sham group there were no changes 0.5h after nicotine injection, 1h later the level of endocannabinoid (2-AG and AEA) began to rise, 2hrs after nicotine injection endocannabinoid content reached a crest value, and backed to its basal level 3hrs after injection. There were no statistical differences between TA group and Sham group(P>0.05)Conclusion: Nicotine increases the content of endocannabinoid(2-AG and AEA) in brain tissue..Experiment 4: Observe the changes of endocannabinoid receptor CB1 in brain after nicotine injectionMethods 56 male SD rats(280-320g), were randomly divided into 7 groups(n=8): Sham group, 1h TA group, 1h Nicotine group, 2h TA group, 2h Nicotine group, 4h TA group, 4h Nicotine group, 6h TA group and 6h Nicotine group. After intraperitoneal injection, the brains of animals were removed in the specified time(1h, 2hrs, 4hrs and 6hrs) to analyze the content of endocannabinoid receptor CB1 in the hemisphere by Western Blot. Take another 9 male SD rats(280-320g), randomly divided into 3 groups(n=3): Sham group, 6h TA group and 6h Nicotine group. The immunofluorescence staining was performed like our previous study[9]. The rats were anesthetized and transcardially perfused with phosphate-buffered saline and 4% paraformaldehyde. The 10-μm thick coronal sections were made for the immunofluorescence staining.Results Six hours after the injection of nicotine hydrogen tartrate salt solution, the content of endocannabinoid receptor CB1 began to rise(P<0.05), there were no statistical differences in other groups(P> 0.05). And in the Sham and TA groups, little expression of CB1 receptor was observed. However, the expression of CB1 receptor was significantly increased in the 6h nicotine groups. Conclusion: Nicotine injection increases the level of endocannabinoid receptor(CB1) in rat brainConclusion:1. Nicotine pretreatment has a protective effect on cerebral I/R injury of rat.2. The neuroprotection of nicotine pretreatment on cerebral I/R injury can be partially reversed by endocannabinoid receptor antagonist AM251.3. Nicotine increases the content of endocannabinoid(2-AG and AEA) in brain tissue..4. Nicotine injection increases the level of endocannabinoid receptor(CB1) in rat brain...